Aptamer's selection using PTEN recombinant protein and PTEN synthetic peptide.

<p><b>A</b>. In these assays, selected oligonucleotides were separated from the GST-PTEN target, indicated above the graph, and quantified by means of real-time PCR. The effect of amplification of the library on the quantity of oligonucleotides obtained is illustrated in this graph. The number of molecules, as indicated on the y axis, is obtained through extrapolation of the standard curve, while the number of cycles applied to the library used is indicated on the x axis. The three assays show a significant increase compared with the library without amplification, used as control. Libraries were amplified through PCR separately for each assay. Standard deviation is indicated using bars on each column. <b>B</b>. Results for assays as described above, using the synthetic peptide of PTEN as target, instead of GST-PTEN. Libraries were separated in aliquots from a larger pool that was amplified through PCR. <b>C</b>. Specificity of the oligonucleotides obtained. Aliquots of the selected oligonucleotides were amplified again through PCR, separated, and split into four dilutions. The filter binding assays were performed with different concentrations for four separate selections of aptamers, and each selection is indicated at the bottom of the graph. The gradient of concentration follows the slope of the triangle and the standard deviation is indicated using bars on each column. <b>D</b>. Affinity of the aptamers obtained. Autoradiography of radioactive aptamers bound to specific targets. The aptamer PTENz7 recognized the recombinant PTEN protein and the peptide without binding to the GST-Anp32e recombinant protein in filter binding assays. <b>E</b>. Selected oligonucleotides developed with 25 cycles of library amplification, using the synthetic peptide of PTEN as target, were cloned into vectors and sequenced. Plasmids from six different clones were labeled by PCR containing ATP-gamma-³⁵PO₄, incubated, and their affinity was measured in Scatchard binding assays. The example shown is the oligonucleotide from clone PTENz7, and the affinity constant measured corresponds to 75 nM, using GST-PTEN protein at a final concentration of 25 µM. At the bottom of the graph the autoradiographic results from the radioactive aptamers retained in the filter binding assays are shown. The concentration of the aptamers used corresponds to the measurements illustrated within the graph. On the x axis, the concentration of PTENz7 in nM is provided. On the y axis, the ratio between bound and free aptamers is shown.